The essential role of ubiquitin in a variety of processes involving protein turnover is now well- established, but central mechanistic features of the multienzyme ubiquitin pathway remain unelucidated. This proposal describes a broad- ranging research program in which engineered ubiquitin fusions will be used to study a number of important features of the ubiquitin pathway, and molecular genetic methods will be used to characterize a number of newly-discovered pathway components. Areas to be addressed include: developing a method to evaluate whether co- translational protein turnover occurs; studies on the mechanics of protein degradation by the proteasome; analysis of the degradation of hybrid proteins containing nearly-identical domains which, when separated, are degraded at divergent rates; a functional and mechanistic analysis of a novel relative of ubiquitin activating enzyme; and a biochemical and genetic analysis of a family of components that are part of a novel `arm' of the ubiquitin pathway. These studies will be carried out mainly in the yeast S. cerevisiae, in which analysis of the ubiquitin pathway is already far advanced.